In multicellular organisms, genomic stability and integrity is maintained by the combined actions of an accurate DNA replication machinery and a complex network of DNA repair pathways. DNA joining is an essential step in DNA replication, in DNA excision repair and in the repair of DNA strand breaks. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Genetic studies have indicated that the LIG4 gene and the functionally homologous DNL4 gene of Saccharomyces cerevisiae participate in the repair of DNA double-strand breaks (DSB)s by non-homologous end joining (NHEJ). In mammalian cells, this pathway is required for genomic stability. Notably, defects in mammalian NHEJ result in the type of genetic rearrangements frequently observed in cancer cells and a predisposition to cancer. In this proposal, we will focus on delineating the molecular mechanisms of DNA ligase IV-dependent NHEJ in S. cerevisiae by a combination of in vitro and in vivo approaches. In published studies we have described physical and functional interactions among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, Pol4, FEN- 1(Rad27) and Dnl4/Lif1 complexes. Preliminary analysis of the assembly of these proteins factors at in vivo DSBs has revealed that Dnl4/Lif1 acts at an unexpectedly early stage of the NHEJ pathway. In Specific Aim 1, we will characterize the complex formed by Hdf1/Hdf2 and Dnl4/Lif1 at DNA ends and elucidate the role of Nej1 in NHEJ. In Specific Aim 2, we will characterize the complex formed by Hdf1/Hdf2, Rad50/Mre11/Xrs2 and Dnl4/Lif1 at DNA ends and determine whether the Hdf1/Hdf2 ring remains topologically linked to DNA following end joining by Hdf1/Hdf2, Rad50/Mre11/Xrs2 and Dnl4/Lif1. Finally, in Specific Aim 3, we will monitor the assembly and disassembly of NHEJ factors during the repair of an in vivo DSB and determine whether the proteasome participates in the turnover of NHEJ complexes assembled at DSB sites. Because of the conservation of NHEJ factors among eukaryotes, the results from our studies will provide insights into NHEJ in mammalian cells and contribute to an overall picture of how the repair of DSBs by NHEJ prevents the deleterious genetic changes associated with cancer cells.